The mechanisms whereby the endosymbiont Wolbachia impacts apoptosis in host cells have been poorly studied. Preferential infection and high accumulation
of Wolbachia in region 2a of the germarium [26] where the checkpoint is located in Drosophila was thought-provoking. We raised the question: Can bacteria Wolbachia in region 2a of the germarium affect the frequency of apoptosis there? Using fluorescence and transmission electron microscopy we compared germaria from ovaries of two D. melanogaster stocks infected with either the wMel or wMelPop strains with germaria from two uninfected counterparts. It was established that the presence of wMel did not alter apoptosis frequency in germaria from D. melanogaster Canton S. In contrast, the number of LDN-193189 germaria containing apoptotic cells in the checkpoint was considerably increased
PF477736 mw in the wMelPop-infected flies as compared with their uninfected counterparts. Thus, evidence was obtained indicating that the virulent Wolbachia strain wMelPop has an effect on the fate of germline cells during D. melanogaster oogenesis. Results Frequency of apoptosis in germaria from ovaries of the uninfected and Wolbachia-infected D. melanogaster Two parts are distinguished in the Drosophila ovariole: the germarium made up of four regions (1, 2a, 2b, 3) and the vitellarium (Figure 1A, B) [27, 28]. The region 2a/2b, where apoptosis can occur, contains 16-cell cysts, somatic stem cells (SSCs), which contact with the somatic stem cell niche (SSCN) and follicle cells (Figure 1B). Cell death in this region of the germarium was detected by two methods, acridine
orange (AO)-staining and TUNEL assay. Fluorescence microscopy of AO-stained ovarioles demonstrated that apoptotic cells were located as large yellow or orange spots in region 2a/2b of the germarium from D. melanogaster (Figure 2A, C, E, G). 3-mercaptopyruvate sulfurtransferase Germaria containing no apoptotic cells fluoresced homogeneous green (Figure 2B, D, F, H). It should be noted that wMel- and wMelPop-infected flies, besides bright spots in region 2a/2b (Figure 2C, G), showed weak punctuate fluorescence both in regions 2a/2b and 1 of the germarium (Figure 2C, D, G, H). Such fluorescent puncta were not observed following TUNEL, thereby indicated that they were not caused by apoptosis. Figure 1 A schematic representation of an ovariole of D. melanogaster . A, an ovariole of D. melanogaster consisting of the germarium (g) and the vitellarium. B, a detailed scheme of the germarium structure composed of regions 1, 2a, 2b, 3. The checkpoint is framed (red). C, a 16-cell cyst; SSCN, a somatic stem cell niche; SSC, a somatic stem cell; FC, a follicle cell. Figure 2 Visualisation of acridine orange (AO)- and TUNEL-stained germarium cells of D. melanogaster . A, C, E, G, germaria containing apoptotic cells in region 2a/2b from 5 day-old uninfected (A, E) and Wolbachia-infected (C, G) females (AO staining).